Synchronous differentiation method of single-sample-derived osteoclast and osteoblast and application of synchronous differentiation method

Abstract

The invention relates to a synchronous differentiation method of single-sample-derived osteoclasts and osteoblasts and application of the synchronous differentiation method, and belongs to the technical field of biology. According to the method, bone marrow cells are extracted from long bones of mammals, adherent cell populations and suspension cell populations are separated after culture, the suspension cell populations are pre-cultured, the adherent cells are differentiated into osteoblasts, and the suspension cells are differentiated into osteoclasts after pre-culture, so that two functional cells are synchronously obtained from the same sample, and the dosage of experimental animals is remarkably reduced. The invention further provides a tartrate-resistant acid phosphatase dye liquor formula and a dyeing method on the basis, the dyeing time is shortened to be within 10 minutes, a high-risk concentrated hydrochloric acid reagent is not needed, heating at 37 DEG C is not needed, operation is easy and convenient, cost is low, and safety is high. According to the scheme, the problems of different sample sources, tedious dyeing process, long period, high cost and the like in the traditional technology are effectively solved, and an efficient and reliable experimental tool is provided for basic researches of bone development, osteoporosis and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for synchronously differentiating single-sample-derived osteoclasts and osteoblasts and application thereof. BACKGROUND

[0002] In the in vitro model of bone development and skeletal disease research, the differentiation culture of osteoblasts and osteoclasts is two core experimental techniques. Bone marrow, as a key cell source, contains mesenchymal stem cells and hematopoietic stem cells, the former can be induced to differentiate into osteoblasts responsible for bone formation, and the latter can differentiate into osteoclasts responsible for bone resorption. These two cells maintain bone metabolism balance in the bone microenvironment, and their in vitro differentiation model has irreplaceable important value for exploring the molecular mechanism affecting bone metabolism and screening drugs for treating bone diseases such as osteoporosis. Therefore, establishing an efficient and reliable differentiation scheme is the key foundation for promoting the research and development of this field.

[0003] However, the existing conventional technical solutions have significant defects. First, since the precursor cells of osteoblasts and osteoclasts need to be sampled from different mouse individuals and cultured independently, it leads to a huge consumption of experimental animals, not only high cost, but also increases the difference between samples, reduces the reliability of experimental data. Secondly, the key technology for identifying osteoclasts, tartrate-resistant acid phosphatase staining method, has obvious drawbacks in its traditional formula or commercial kit: complicated operation steps, a large number of reagents required, difficulty in self-preparation, high overall cost, and the need to use hydrochloric acid and other high-risk corrosive reagents, which pose a threat to the safety of experimental personnel. In addition, the differentiation culture cycle is long, and the consumption of expensive cytokines further increases the experimental cost. Finally, the traditional tartrate-resistant acid phosphatase staining reaction process takes as long as 1 hour, requires heating at 37℃, and is low in efficiency, which becomes a bottleneck limiting the experimental throughput. These problems jointly restrict the efficiency and scale of related research. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for synchronously differentiating single-sample-derived osteoclasts and osteoblasts, which extracts bone marrow cells from mammalian long bones, separates adherent cell groups and suspension cell groups after culture, and pre-cultures the suspension cell groups. The adherent cell groups are induced to differentiate into osteoblasts, and the suspension cell groups are differentiated into osteoclasts, achieving the synchronous acquisition of the two functional cells from the same sample. On this basis, the present application provides a new tartrate-resistant acid phosphatase staining solution formula and a staining method, which shortens the staining time to less than 10 minutes and does not require heating at 37℃.

[0005] The first object of the present application is to provide a method for synchronously differentiating single-sample-derived osteoclasts and osteoblasts, which comprises the following steps: Step S1, obtaining a bone marrow cell suspension from a mammal long bone; Step S2, culturing the bone marrow cell suspension with complete medium to obtain a first adherent cell population and a suspension cell population, resuspending the first adherent cell population after digestion with complete medium added with an inducing agent, and obtaining osteoblasts after differentiation; Step S3, pre-culturing the suspension cell population to obtain a second adherent cell population, using a medium mixed at a ratio of complete medium and macrophage colony-stimulating factor for the pre-culturing, adding macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand after digestion of the second adherent cell population, and obtaining osteoclasts after differentiation.

[0006] Further, the long bone is one or several of humerus, femur and tibia.

[0007] Further, the bone marrow cells need to be cultured for 4-5 days.

[0008] Further, the pre-culturing is performed for 1-2 days.

[0009] Further, the inducing agent contains β-glycerophosphate and ascorbic acid.

[0010] In an embodiment of the present application, the inducing agent contains 10 mM of β-glycerophosphate and 50 μg / mL of ascorbic acid.

[0011] Further, the concentration of the macrophage colony-stimulating factor and the receptor activator of nuclear factor kappa B ligand is 30-50 ng / mL.

[0012] In an embodiment of the present application, the concentration of the macrophage colony-stimulating factor is 40 ng / mL.

[0013] In an embodiment of the present application, the concentration of the receptor activator of nuclear factor kappa B ligand is 40 ng / mL.

[0014] Further, the digestion step of the second adherent cell population is: discarding the culture medium, washing twice with phosphate buffer, adding trypsin digestion solution for 5 minutes, adding complete medium to terminate digestion, centrifuging the cell suspension, discarding the supernatant, and resuspending with culture medium.

[0015] Further, the buffer is phosphate buffered saline solution.

[0016] Further, the amount of trypsin digestion solution added is 1000 μL.

[0017] Further, the digestion condition is 37℃, and the digestion time is 5 minutes.

[0018] Further, the centrifugal condition is 1000 rounds per minute, and the centrifugal time is 5 minutes.

[0019] A second object of the present application is to provide the use of the above-mentioned synchronized differentiation method in screening drugs for treating bone diseases.

[0020] Further, the method further comprises a step of identifying the osteoclasts by using tartrate-resistant acid phosphatase staining solution, wherein the components of the tartrate-resistant acid phosphatase staining solution comprise naphthol AS-MX phosphate, dimethylformamide, acetic acid, sodium acetate, and fast violet LB salt.

[0021] Further, the step comprises removing the cell culture medium, washing with phosphate buffer solution, adding polyformaldehyde for fixation, removing the polyformaldehyde, adding 1% triton X-100 for washing, removing the triton X-100, and then adding the tartrate-resistant acid phosphatase staining solution.

[0022] Further, the staining time is less than 10 minutes.

[0023] In one embodiment of the present application, the polyformaldehyde fixation time is 10 minutes.

[0024] In one embodiment of the present application, the concentration of the polyformaldehyde is 40 g / L.

[0025] In one embodiment of the present application, the amount of the polyformaldehyde added is 200 μL.

[0026] A third object of the present application is to provide a tartrate-resistant acid phosphatase staining solution, wherein the components of the tartrate-resistant acid phosphatase staining solution comprise naphthol AS-MX phosphate, dimethylformamide, acetic acid, sodium acetate, and fast violet LB salt.

[0027] Further, the concentration of the naphthol AS-MX phosphate is 0.1-0.2 mg / mL, the concentration of the dimethylformamide is 10-12 μL / mL, the concentration of the acetic acid is 0.2-0.3 mol / L, and the concentration of the sodium acetate is 0.2-0.3 mol / L.

[0028] In one embodiment of the present application, the tartrate-resistant acid phosphatase staining solution is a 10 mL system, which comprises 1 mg of naphthol AS-MX phosphate, 100 μL of dimethylformamide, 3 mL of 0.2 mol / L acetic acid, 7 mL of 0.2 mol / L sodium acetate, and 6 mg of fast violet LB salt.

[0029] A fourth object of the present application is to provide the use of the above-mentioned tartrate-resistant acid phosphatase staining solution in identifying osteoclasts.

[0030] The present application has the following beneficial effects: The simultaneous differentiation method provided by this invention utilizes a single sample to simultaneously obtain two cell types, reducing animal usage by more than 50%, complying with animal ethics and lowering experimental costs. Homologous sample differentiation avoids batch-to-batch variations, making comparative experimental data on osteoblasts and osteoclasts more reliable and meaningful. The pre-culture step further enhances osteoclast differentiation and increases cell purity. Furthermore, this invention provides a novel tartrate-resistant acid phosphatase staining formula, reducing staining time from 1 hour to less than 10 minutes, eliminating the need for 37°C heating, greatly simplifying the process, and avoiding the use of hazardous reagents such as concentrated hydrochloric acid, making it safer and more economical. Combining the simultaneous differentiation method with rapid staining technology forms a complete, efficient, and lower-cost solution for in vitro differentiation research of osteoblasts and osteoclasts. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 The results of osteoblast alkaline phosphatase staining are shown in the embodiments of the present invention. Figure 2 The results of silver nitrate staining of osteoblasts are shown in the embodiments of the present invention. Figure 3 The results of tartrate-resistant acid phosphatase staining of osteoclasts are shown in the embodiments of the present invention. Figure 4 The above are the group I osteoclast staining results in this embodiment of the invention, where A is the staining result of the kit and B is the staining result of the staining solution; Figure 5 The above are the staining results of osteoclasts in Group II of this invention, where A is the staining result of the kit and B is the staining result of the staining solution. Figure 6 The above are the staining results of osteoclasts in Group III of this embodiment, where A is the staining result of the kit and B is the staining result of the staining solution. Figure 7 These are osteoclasts obtained from Group I and Group III in the embodiments of the present invention, wherein A is osteoclasts from Group I and B is osteoclasts from Group III; Figure 8 These are osteoblasts obtained from groups III and IV in the embodiments of the present invention, wherein A is osteoblasts from group III and B is osteoblasts from group IV. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] Example

[0034] One integrated cell acquisition (1) Take six long bones of a mouse, including humerus, femur and tibia, and place them in a container containing medium (containing double antibodies) after removing the muscles for standby; (2) Prepare a clean 20 mL syringe, needle, scissors and tweezers in advance, and move them into the clean bench after sterilization. The syringe is used to suck 20 mL of complete medium containing fetal bovine serum, and the two ends of each long bone are cut off. The bone marrow is flushed into a 50 mL centrifuge tube with a syringe, and attention is paid to collect as much bone marrow as possible; (3) Mix the collected bone marrow-containing medium mixture and blow it evenly on two 10 cm diameter cell culture dishes, each containing 10 mL of complete medium; (4) Add 5 mL of complete medium every two days in the culture dish, and culture at 37°C, 5% CO2 for 4-5 days. When the cells adhere to about 30% of the total area of the cell culture dish, the next step can be performed. At this time, the adherent cells are mesenchymal stem cells and can be used for subsequent experiments; (5) Collect the culture medium and non-adherent bone marrow cells in a 10 cm diameter cell culture dish in a 50 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Add 10 mL of complete medium to each of the original cell culture dishes, and culture at 37°C, 5% CO2 for 1-2 days before use for osteoblast differentiation experiments.

[0035] Two, pre-culture of osteoclasts Centrifuge to remove supernatant, and the remaining system contains hematopoietic stem cells. Add 4 mL of complete medium, mix evenly by blowing, add macrophage colony-stimulating factor M-CSF (concentration of 40 ng / mL), mix evenly by blowing, and place in a six-well plate, 2 mL per well, for pre-culture. Culture at 37°C, 5% CO2 for 1-2 days, and the adherent cells can be used for osteoclast differentiation experiments. When osteoclasts are obtained alone, pre-culture with bone marrow suspension.

[0036] Three, osteoblast differentiation Discard the culture medium of the obtained adherent cells, rinse with phosphate buffer twice, add 400 μL of trypsin digestion solution, and digest at 37°C for 5 minutes. Add complete medium to terminate digestion, and collect into a 50 mL centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, discard the supernatant, resuspend for counting with medium, and place in a 48-well plate, 1.0×10 5 cells per well. The next day, add 10 mM β-glycerophosphate and 50 μg / mL ascorbic acid to start differentiation, and change the differentiation medium every three days. It is expected that 4-6 days can be used for alkaline phosphatase staining, and the staining results are shown in Figure 1 . 7-9 days can be used for silver nitrate staining, and the results are shown in Figure 2 .

[0037] Four, osteoclast differentiation After the pre-cultured adherent cells were discarded, the medium was washed with phosphate buffer, 200 μL of trypsin digestion solution was added, and it was digested at 37°C for 5 minutes. The digestion was terminated by adding complete medium, and it was collected into a 50 mL centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the osteoclast differentiation medium (containing 90% medium, 10% serum, 40 ng / mL macrophage colony-stimulating factor, and 40 ng / mL receptor activator of nuclear factor kappa B ligand) was resuspended and counted in a 48-well plate at 4.6 x 10 4

[0038] Five, optimization of tartrate-resistant acid phosphatase staining The tartrate-resistant acid phosphatase staining solution provided in this embodiment has the following formulation: for a 10 mL system, it contains 1 mg of naphthol AS-MX phosphate, 100 μL of dimethylformamide, 3 mL of 0.2 M acetic acid, 7 mL of 0.2 M sodium acetate, and 6 mg of fast violet LB salt.

[0039] The optimized tartrate-resistant acid phosphatase staining method provided in this embodiment is as follows: (1) Take the differentiated cells, remove the medium, and wash twice with phosphate buffer, add 200 μL of 4% paraformaldehyde per well for 10 minutes of fixation; (2) Remove the paraformaldehyde and wash with pure water; (3) Wash once quickly with phosphate buffer containing 1% triton; (4) Add the above-mentioned tartrate-resistant acid phosphatase staining solution, and the staining time is 5-10 minutes; (5) Wash 2-3 times with pure water, dry, and take a photo to determine the differentiation of osteoclasts. The staining results of the osteoclasts obtained in step three are shown in Figure 3

[0040] To further verify the effect of the simultaneous differentiation method and staining method provided in this embodiment, the long bone marrow of wild-type mice was extracted according to the above method and divided into four groups on average: Group I used the bone marrow suspension directly for the pre-culture step of osteoclasts, and after pre-culture, it was used for the differentiation of osteoclasts, group II was cultured according to the kit instructions, group III integrated the culture and differentiation of osteoclasts and osteoblasts, and group IV only cultured osteoblasts. The bone marrow was collected and mixed uniformly by blowing after washing with complete medium containing serum, and it was plated in a 10 cm diameter cell culture dish. The cells adhered in 4-5 days, and 5 mL of complete medium was supplemented every two days during the period.

[0041] ​​I group is added with macrophage colony stimulating factor M-CSF (concentration is 40 ng / mL) in bone marrow suspension, and is pre-cultured in a six-well plate, 2 mL per well, and is cultured at 37℃, 5% CO2 for 1-2 days, and after pre-culture, differentiation of osteoclasts is carried out, one group is dyed by using a tartrate-resistant acid phosphatase staining kit, and is dyed at 37℃ for 1 hour, and the dyeing result is as shown in A of Figure 4 , one group is dyed by using the tartrate-resistant acid phosphatase staining solution and the optimized dyeing method provided in the embodiment, and the dyeing time is 10 minutes, and the dyeing result is as shown in B of Figure 4 .

[0042] II group is cultured according to the steps of the tartrate-resistant acid phosphatase staining kit instructions, one group is dyed by using the tartrate-resistant acid phosphatase staining kit, and is dyed at 37℃ for 1 hour, and the dyeing result is as shown in A of Figure 5 , and the other group is dyed by using the tartrate-resistant acid phosphatase staining solution and the optimized dyeing method provided in the embodiment, and the dyeing time is 10 minutes, and the dyeing result is as shown in B of Figure 5 .

[0043] III group is integrally cultured and differentiated with osteoclasts and osteoblasts, one group is dyed by using the tartrate-resistant acid phosphatase staining kit, and is dyed at 37℃ for 1 hour, and the dyeing result is as shown in A of Figure 6 , and the other group is dyed by using the tartrate-resistant acid phosphatase staining solution and the optimized dyeing method provided in the embodiment, and the dyeing time is 10 minutes, and the dyeing result is as shown in B of Figure 6 .

[0044] The comparison between I group and III group is as shown in Figure 7 , and it can be seen that the synchronous acquisition scheme does not affect the number of osteoclasts, and the comparison between III group and IV group is as shown in Figure 8 , and it can be seen that the synchronous acquisition scheme does not affect the number of osteoblasts.

[0045] From the above results, it can be seen that the differentiation degree of the pre-cultured I group osteoclasts is higher than that of the II group without pre-culture, and the counting is more convenient when plating, and the cell state is better. The III group integrally cultured and differentiated needs to wait for 4 days for the mesenchymal stem cells to adhere, but does not affect the state of the osteoclasts, and since the cells are adhered and then aspirated to plate new ones, the cell state of the III group is also better than that of the I group, and there are fewer impurities brought by the primary cells. The dyeing results of each treatment group prove that by using the dyeing solution and the dyeing method provided in the embodiment, the dyeing time is shortened to within 10 minutes, without 37℃ heating, and the coloring effect is significantly enhanced.

[0046] Comparative Example I. Preparation of osteoclast differentiation (1) Take the mouse femur and tibia, cut off both ends of the joint, and store them in a sterile cell culture dish and move to the biological safety cabinet. Use a 10 mL syringe to suck 10 mL of phosphate buffer, rinse the bone marrow into a 50 mL sterile tube, and centrifuge at 1400 rpm for 5 minutes; (2) Discard the supernatant, resuspend the cells, move them to a 10 cm cell culture dish, and incubate overnight at 37°C, 7% CO2 cell incubator; (3) The next day, collect the cells and centrifuge at 1400 rpm for 5 minutes, discard the supernatant, add 10 mL of phosphate buffer and centrifuge at 1400 rpm for 5 minutes; (4) Discard the supernatant, resuspend the cells with 10 mL of medium and count, adjust the cell number to 1.0 x 10 5 cells / mL using medium. For a 24-well plate, add 20 μL of macrophage colony-stimulating factor (concentration: 2 ng / μL) and 1.0 x 10 5 cells per well and incubate overnight at 37°C, 7% CO2; (5) The next day, add 1 μL of nuclear factor kappa B receptor activator ligand (concentration 40 ng / μL) to each well, then incubate at 37°C, 7% CO2 cell incubator for 3 days; (6) Change the medium, add 20 μL of macrophage colony-stimulating factor and 1 μL of nuclear factor kappa B receptor activator ligand to each well, and incubate at 37°C, 7% CO2 cell incubator. Change the differentiation medium every 3 days to obtain osteoclasts.

[0047] II. Preparation of osteoblast differentiation The mouse femur and tibia bone marrow was collected with medium and plated on a 10 cm diameter cell culture plate, cultured for 4-7 days until the cells adhered, then the medium was changed and cultured for 1-2 days. The adherent cells obtained can be used for osteoblast differentiation experiments.

[0048] Using the method provided in the present comparative example, osteoblast and osteoclast differentiation was carried out separately, which required separate sampling of mice, consumed a large number of mice, and had a long differentiation period, consumed a large amount of differentiation reagent, increased the cost of osteoblast and osteoclast differentiation preparation, and introduced differences between samples, the preparation period of osteoblasts and osteoclasts was significantly different, which reduced the comparability and reliability of experimental data.

[0049] Obviously, the above embodiments are merely example for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and can not be exhausted. The obvious changes or variations derived therefrom are still within the scope of the present invention.

Claims

1. A method for the simultaneous differentiation of osteoclasts and osteoblasts from a single sample, characterized in that, The synchronous differentiation method includes the following steps: Step S1: Obtain bone marrow cell suspension from mammalian long bones; Step S2: Culture the above bone marrow cell suspension in complete culture medium to obtain the first adherent cell population and the suspension cell population. After digestion, the first adherent cell population is resuspended in complete culture medium with added inducing agent and differentiated to obtain osteoblasts. Step S3: The above-mentioned suspended cell population is pre-cultured to obtain a second adherent cell population. The culture medium used for pre-culture is a mixture of complete culture medium and macrophage colony-stimulating factor. After digestion, macrophage colony-stimulating factor and nuclear factor κB receptor activator ligand are added to the second adherent cell population to differentiate into osteoclasts.

2. The synchronous differentiation method according to claim 1, characterized in that: The inducer contains β-glycerophosphate and ascorbic acid.

3. The synchronous differentiation method according to claim 1, characterized in that: The concentrations of the macrophage colony-stimulating factor and the nuclear factor κB receptor activating factor ligand are 30-50 ng / mL.

4. The synchronous differentiation method according to claim 1, characterized in that: The digestion steps include discarding the culture medium, rinsing with phosphate buffer, adding trypsin digestion solution, digesting for 5 minutes, adding complete culture medium to terminate digestion, centrifuging, discarding the supernatant, and resuspending in culture medium.

5. The application of the synchronous differentiation method according to any one of claims 1-4 in screening drugs for the treatment of bone diseases.

6. The application according to claim 5, characterized in that: It also includes the step of identifying osteoclasts using a tartrate-resistant acid phosphatase staining solution, the components of which include naphthol AS-MX phosphate, dimethylformamide, acetic acid, sodium acetate, and solid red violet LB salt.

7. The application according to claim 6, characterized in that: The steps include removing the cell culture medium, washing with phosphate buffer, fixing with paraformaldehyde, removing the paraformaldehyde, washing and adding 1% Triton solution, and then adding the tartrate-resistant acid phosphatase staining solution.

8. A tartrate-resistant acid phosphatase staining solution, characterized in that: The components of the tartrate-resistant acid phosphatase staining solution include naphthol AS-MX phosphate, dimethylformamide, acetic acid, sodium acetate, and solid red violet LB salt.

9. The tartrate-resistant acid phosphatase staining solution according to claim 8, characterized in that: The concentration of naphthol AS-MX phosphate is 0.1-0.2 mg / mL, the concentration of dimethylformamide is 10-12 μL / mL, the concentration of acetic acid is 0.2-0.3 mol / L, and the concentration of sodium acetate is 0.2-0.3 mol / L.

10. The application of the tartrate-resistant acid phosphatase staining solution according to claim 8 or 9 in osteoclast identification.

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